The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide.

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (approximately 98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (approximately 76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (98.5 percent) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (76 percent) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V

Cryofibrinogenaemia: a study of 49 patients.

The purpose of this study was to characterize the clinical features and components of 30 patients with isolated cryofibrinogen (CF) versus those of 19 patients with combined CF and cryoglobulins (CG). Secondary forms of cryofibrinogenaemia associated with collagen disorders, infectious or malignant diseases, were significantly more frequent in patients with combined CF and CG than those with isolated CF (79 versus 47%, P = 0.02). Both groups of CF patients presented predominantly cutaneous symptoms (77% in isolated CF; 58% in combined CF + CG), and less frequently venous and/or arterial thrombosis (13% in isolated CF; 3% in combined CF + CG). Patients with idiopathic forms of CF, and particularly those without CG, suffered essentially from recurrent painful skin ulcers, mainly triggered by cold exposure. Patients with isolated CF had higher mean plasma concentrations of CF than those with combined CF + CG (1. 61 +/- 1.26 versus 0.82 +/- 1.18 g/l, respectively; P = 0.004), but there was no correlation between the CF plasma level and either the severity of symptoms or the sensitivity to cold. In patients with isolated CF, fibronectin was suggested (by precipitation analysis) to be a major component of the cryoprecipitate, whereas immunoglobulins were rarely present (in only three out of 30 patients). By contrast, in the majority of patients (78%) with combined CF and CG, the CF consisted mainly of immunoglobulins of the same class as those characterizing the associated CG. Analysis of the CG precipitate revealed the presence of fibronectin but not fibrinogen, alpha1-antitrypsin and alpha2-macroglobulin. In conclusion, isolated and combined cryofibrinogenaemia are associated with different clinical signs requiring different clinical management, but there is no evidence as yet for a causal role of the cryoprecipitates in the differences observed.

Effect of Shiga toxin 2 on water and ion transport in human colon in vitro.

Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium.

Molecular and functional characterization of an amphibian urea transporter.

We report the characterization of a frog (Rana esculenta) urea transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box urea transporter signature sequence were found in this amphibian urea transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thiourea, but the fUT-mediated urea transport was strongly inhibited by this compound. Thus, this amphibian urea transporter displays transport characteristics in between those of UT-A2 and UT-B.

Functional characterization and localization of AQP3 in the human colon.

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98 percent identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells

Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo.

The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine. The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection. These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage. We describe here a test capable of determining the diarrheagenic potential of attenuated V. cholerae strains. The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo. We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V. cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.

Tennis after total hip arthroplasty.

The purpose of this study was to characterize patients who play tennis after undergoing hip arthroplasty in terms of their functional abilities and degree of satisfaction. A questionnaire was sent to all United States Tennis Association member associations in a tri-state area to identify players who had undergone a hip arthroplasty. The study group had 50 men and 8 women with a mean age of 70 years (range, 47 to 89). Only 14% of the patients' surgeons approved this tennis activity, with 34% of the surgeons recommending only doubles. Three patients required revision surgery after a mean of 8 years. One year after arthroplasty, players played both singles and doubles approximately three times per week. All tennis players were extremely satisfied with their hip arthroplasties and their increased ability to participate in their favorite sport. Because this study was confined to association members, further studies are needed to assess the general effect of tennis on total hip arthroplasty. Until future studies are performed, the authors would recommend that physicians advise caution in tennis activities and to carefully follow their patients yearly to see if osteolysis is occurring prematurely.

The inhibition of the reverse transcriptase of HIV-1 by the natural sulfoglycolipids from cyanobacteria: contribution of different moieties to their high potency.

The potent in vitro inhibition of the enzymatic activity of the human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) by the lipophilic extracts of cyanobacteria8 was primarily attributed to the sulfoquinovosylpranosyl lipids, compounds 1-4. These sulfolipids inhibit efficiently and selectively only the DNA polymerase activity of HIV-1 RT (and not the ribonuclease H function) with 50% inhibitory concentration value (IC50) as low as 24 nM exhibited by compound 1. The novel natural compound 4, in which two hydroxy groups on the sugar moiety are substituted by palmitoyl residues, exhibits a significant decrease in the maximal inhibition capacity. It is possible, therefore, that the contribution of acylated groups to the molecule at these positions interferes with inhibition, possibly, by steric hindrance. Both the sulfonic acid moiety and the fatty acid ester side chain have a substantial effect in potentiating the extent of inhibition. For one, the inhibitory effects of all the natural glycolipids tested (5-8) are markedly reduced, and the hydrolysis of the fatty acid side chain, as in derivative 9, has substantially abolished the inhibition of HIV RT.

Treatment of grade III acromioclavicular separations in professional throwing athletes: results of a survey.

Forty-two team orthopedists representing all 28 major league baseball teams were surveyed to ascertain their definitive treatment for a hypothetical starting rotation pitcher who had sustained a grade III acromioclavicular (AC) separation to his throwing arm 1 week before the season. Twenty-nine (69%) of the physicians would treat the injury nonoperatively, while 13 (31%) would operate immediately. Twenty-five (60%) of the orthopedists had actually treated a pitcher or position baseball player with a grade III AC separation in the throwing arm, the 25 treating a total of 32 patients. Twenty (63%) of these injuries were treated nonoperatively, and 12 (37%) were treated operatively. The physicians reported that 16 (80%) of the patients treated nonoperatively regained normal function and achieved complete relief of pain, while 18 (90%) had normal range of motion after treatment; of those treated operatively, 11 (92%) regained normal function, achieved complete relief of pain, and had normal range of motion after surgery.

Soleus neurectomy for dynamic ankle equinus in children with cerebral palsy.

Problems with the gastrocnemius-soleus muscle group can severely impair the gait of children with cerebral palsy. Treatments, including bracing, muscle lengthening, neurectomy, or a combination, have been used with mixed results. Soleus neurectomy was performed as the primary treatment for ankle clonus in 38 legs of 21 children with a variety of cerebral palsies. Concurrent heel cord or muscle lengthening was performed if needed. Patients were followed for an average of 9 years (range, 2 to 14 years). Clonus recurred in 4 treated ankles. In 2 cases, this was due to a nerve anomaly. Postneurectomy Achilles tendon lengthening was required in 8 of the treated ankles. Neurectomy was beneficial for 19 of 21 children. Functional improvements included better control of stopping, better balance, and less toe walking. The greatest improvement was seen in those patients who did not also have heel cord contractures.

The retroisthmic cleft. Scintigraphic appearance and clinical relevance in patients with low back pain.

This article describes the scintigraphic appearance of both a symptomatic and asymptomatic retroisthmic cleft in two athletes with low back pain. This lesion, which involves the lamina, is the least common of the neural arch defects of which spondylolysis is the most common. The anatomy of the lesion is discussed and illustrated. The literature about the possible cause of these lesions is reviewed. The report emphasizes the valuable role of radionuclide bone imaging in patients who have pain of potentially osseous origin, and who have a lesion of uncertain physiologic significance seen on an anatomic study such as a plain x-ray, CT scan, or MRI.

New acylated sulfoglycolipids and digalactolipids and related known glycolipids from cyanobacteria with a potential to inhibit the reverse transcriptase of HIV-1.

Five novel diacylated sulfoglycolipids (1-5) were isolated from the cyanobacterium Scytonema sp. (TAU strain SL-30-1-4) and four novel acylated diglycolipids (6-9) were isolated from the cyanobacterium Oscillatoria raoi (TAU strain IL-76-1-2). These two groups of glycolipids and related known glycolipids isolated from these two and three other strains of cyanobacteria, Phormidium tenue (TAU strain IL-144-1), O. trichoides (TAU strain IL-104-3-2), and O. limnetica (TAU strain NG-4-1-2), were found to inhibit HIV-1 RT enzymatic activity to different extents. The structure elucidation of the various compounds is based on the selective hydrolysis of the glycerol ester moieties, GCMS analysis of the methyl ester derivatives of the liberated fatty acids, homo- and heteronuclear-2D-NMR techniques, and MS. The use of negative-ion FABMS for analyzing the combination and distribution of the fatty acids in glycolipids is demonstrated.

[Migration and professions: deducing Italian immigration to Argentina from nominative sources]. / Migracion y profesiones: una lectura del movimiento inmigratorio italiano a la Argentina desde las fuentes nominativas.

PIP: "The article examines the possibilities of systematically exploiting nominative document corpuses related to immigration in Buenos Aires [Argentina] in the period of mass migration [1885-1910], and proposes to focus on the occupations declared by the Italian immigrants. After reviewing the formal aspects of the documental series used (missing information, polysemy of professional aggregates) the immigration of masons is analyzed, taking into account demographic characteristics, annual rhythms of arrival, [and] migration typologies." (EXCERPT)^ieng

A modified therapeutic community for the dually diagnosed. Greenhouse Program at Bellevue Hospital.

Techniques used in therapeutic communities may be applicable to patients dually diagnosed with mental illness and a psychoactive substance use disorder (PSUD). This study was designed to evaluate the demographics, course, and outcome for 100 patients treated in one such residential program. One hundred indigent male patients admitted to a drug-free therapeutic community for the dually diagnosed were studied on admission and over the course of their treatment, and subjects were monitored throughout their stays on the basis of observed urine toxicology tests and a clinical assessment of drug or alcohol use. The mean age of the patients was 33.8 years, and the average length of stay was 121.0 days. Thirty-three of the patients completed the full 6-month program and moved on to another stable living environment. Only 12 patients had urine toxicologies positive for illicit drugs or alcohol while in the program. These findings support the possibility of applying the residential drug-free therapeutic community to dually diagnosed patients.

The MICA therapeutic community: challenges for residents and staff.

Therapeutic communities (TCs) are residential self-help programs for recovering addicts. Traditional TCs are staffed by recovered addicts. Recently the TC model has been modified for the treatment of chemical abusers with severe mental illness. These programs are to varying degrees staffed by mental health professionals. Furthermore, they are often housed in institutions whose model for care is based on service to patients rather than self-help. There are inherent conflicts in the modified TC which is a hybrid of these two treatment approaches. This paper explores issues related to the roles of patients, mental health professionals, and the parent institution in the TC modified for the treatment of the mentally ill chemical abuser.